Method for the detection of bacterial species of the  genera  anaplasma/ehrlichia and bartonella

ABSTRACT

The present invention relates to a method for the detection and identification of bacterial species belonging to the genera  Anaplasma/Ehrlichia  and  Bartonella , and also provides triggers and probes required for its application, as well as associated kits.

This application claims the benefit of priority to Spanish Patent Application No. ES 200701830, filed Jun. 29, 2007, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a method for the detection and identification of bacterial species belonging to the genera Anaplasma/Ehrlichia and Bartonella (ES 2264642), and also provides triggers and probes required for its application, as well as associated kits.

BACKGROUND

To date, about 200 zoonotic diseases (e.g., bartonelosis, leptospirosis, Lyme borreliosis, etc.), which affect humans and represent one of the main causes of death and entail substantial economic loss in third world countries, have been described. Coexistence with animals, lack of sanitary infrastructure and low cultural level continue to be the main allies of these diseases.

In the same manner, certain types of zoonosis which are widespread in third world countries are now thriving in industrialized countries as a consequence of population increases in urban and periurban areas, and increased movement of animals across international borders. These circumstances, amongst others, entail the risk of introducing exotic diseases into the environment.

Additionally, the frequent findings of arthropods infected by more than one of the pathogens included in the present invention, increases the possibility of more than one zoonotic disease being transmitted in a single bite. As a result, hospitalizations due to medical profiles produced by contact with animals or arthropods, such as mosquitoes, ticks, fleas, lice, mites, etc., which act as vectors or pathogen reservoirs, is becoming increasingly common. Said medical profiles, due to their high degree of similarity, do not allow a fast and reliable identification of the pathogenous agent, so that specific and fast treatment is not possible and is occasionally administered too late. This undoubtedly justifies the need for a comprehensive method to detect and identify bacterial species that cause zoonosis.

To date, the molecular diagnosis methods available are basically limited to the detection of pathogens based on antibody technology. This type of analysis, generally retrospective and with low sensitivity levels, is normally of little use to treating diseases in acute-phase states.

Another alternative for the detection and identification of pathogens is based on the application of culture mediums. These types of techniques are scarcely applicable to certain species of genera, such as Bartonella and Anaplasma/Ehrlichia, due to the fact that said species do not normally grow in regular culture mediums and may even require cellular cultures. As a result, these methodologies are isolated from regular practice in hospital microbiology laboratories. One of the most effective alternatives to these types of methodologies is the direct analysis of genetic material, based on Polymerase Chain Reaction (PCR) technology. Said technology, while being highly effective, is greatly limited by the difficulty in finding specific markers or regions, in addition to triggers and probes, which ensure reliable sample analysis.

A paper has recently been published (Blaskovic D. et al. 2005. FEMS Microbiology Letters 243:273-8) which describes a method based on ribosomal DNA analysis, although it uses universal triggers which amplify the genetic material of both target and non-target bacteria, due to which its sensitivity is substantially reduced.

Other methods, such as those described by U.S. Pat. Nos. 6,300,072 and 6,518,020, are capable of detecting and identifying bacteria of the genus Bartonella by using the same region (16S-23S), even though the number of species within this genus has increased substantially since said patents were filed and their approximation, which consists of discriminating between species according to the size of the amplicon obtained during PCR, is not useful for certain known species within the same genus which are similar in size to the amplified fragment.

SUMMARY OF THE INVENTION

The present invention relates to the use of genes 16S and msp2 and intergenic space 16S-23S for the detection and identification of the different bacterial species and groups of species that cause zoonosis, improving the previously described procedures based on its capacity to specifically detect a substantial number of bacterial species using probes and triggers with high sensitivity levels.

Therefore, the present invention solves the problem of the tediousness and complexity of detecting a high number of bacteria that cause zoonosis which can be clinically and/or epidemiologically indistinguishable, through the development of a detection method and Kit based on PCR technology. Specifically, the invention allows the analysis of different regions of bacterial DNA belonging to the genera Anaplasma/Ehrlichia and Bartonella in order to identify both genus and species, as shown in the following table (Table 1).

In most cases, the species identified correspond to cultured species and, in others, correspond to species isolated for the first time (non-cultured species). Said species have been obtained from samples of Meles meles (badger), Ixodes ricinus and Apodemus sylvaticus species and have been characterized by the sequences AJ269792 and SEQ ID NO:44-46, as shown in Table 3.

TABLE 1 BACTERIAL SPECIES GENE Anaplasma/Ehrlichia A. phagocytophilum msp2 msp2 Ehrlichia ruminantium 16S E. sennetsu 16S E. risticii 16S E. muris 16S A. platys 16S E. canis/E. ovina 16S Bartonella Generic 16S B. talpae 16S-23S Bartonella sp.* 16S-23S B. phoceensis 16S-23S B. rattimasiliensis 16S-23S Bartonella sp. detected in Apodemus sylvaticus 16S-23S B. rochalimae 16S-23S Bartonella sp. detected in badger 16S-23S Bartonella sp** detected in Ixodes ricinus 16S-23S Bartonella sp detected in Ixodes ricinus 16S-23S *B. chomeli/schoenbuchensis/capreoli/birtlesii

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. This figure represents two hybridization membranes for the validation of triggers and probes used to detect the Bartonella genus. In the membrane on the left, the absence of crossed reactivity between the different probes within the genus is shown. In the membrane on the right, the absence of crossed reactivity in samples which do not contain the Bartonella genus is shown. The S-Cl2 probe refers to the Internal Amplification Control (IAC) probe.

FIG. 2. This figure represents two hybridization membranes for the validation of triggers and probes used to detect the genera Anaplasma/Ehrlichia. In the membrane on the left, the absence of crossed reactivity between the different probes within the genus is shown. In the membrane on the right, the absence of crossed reactivity in samples which do not contain the genera Anaplasma/Ehrlichia is shown. The S-Cl2 probe refers to the IAC probe.

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect of the invention, said invention relates to a method (hereinafter, method of the invention) for the detection and identification, preferably simultaneous, of any of the bacterial species and genera, as indicated in Tables 2 and 3, comprising the following steps:

-   -   a. Amplifying any of the sequences related to the group         comprised of SEQ ID NO:1-17, 47 and/or their complementary         sequences using specific triggers.     -   b. Detecting the amplification of the sequences mentioned in         step a), said amplification being indicative of the presence or         absence of the bacterial genera or species that cause zoonosis,         as indicated in Tables 2 and 3.

The triggers required to apply the method of the invention may be designed by means of multiple alignment with the sequences comprising SEQ ID NO:1-17 and 47 using computer programs such as CLUSTAL X, and allow the identification of highly preserved regions that will act as a mould for trigger design, which must subsequently be validated empirically.

According to a preferred embodiment of this aspect of the invention, the triggers are capable of hybridizing with different nucleotide regions of the genes 16S, msp2 and intergenic space 16S-23S (Tables 2 and 3), although the sequences of said triggers shall preferably be selected from the SEQ ID NO:18-25 group and/or their complementary sequences, these being capable of amplifying SEQ ID NO:1-17, 47 and/or their complementary sequences, in a preferably simultaneous manner. These triggers, in addition to simplifying the method, have the advantage of low or null reactivity with respect to samples of other species (see Table 5).

According to an even more preferred embodiment of this aspect of the invention, the detection of sequences SEQ ID NO:1-17, 47 and/or their complementary sequences may be carried out based on well-known methodologies within the art, preferably using probes. According to an even more preferred embodiment, said probes are capable of hybridizing between the positions of genes 16S, msp2 and intergenic space 16S-23S, as indicated in Tables 2 and 3, although said probes will preferably comprise the sequences selected from the group that comprises SEQ ID NO:26-42, 48 and/or their complementary sequences.

A second aspect of the invention relates to triggers capable of amplifying the sequences selected from the group comprising SEQ ID NO:1-17, 47 and their complementary sequences. Preferably, said triggers shall be capable of hybridizing between the nucleotide positions of genes 16S, msp2 and intergenic space 16S-23S, as indicated in Tables 2 and 3 (column 3). According to an even more preferred embodiment, the triggers comprise the sequences selected from the group SEQ ID NO:18-25 and/or their complementary sequences. Hereinafter, these will be referred to as triggers of the invention.

A third aspect of the invention relates to probes capable of specifically detecting any of the bacterial species and genera, as indicated in Tables 2 and 3 (column 6), said probes being capable of hybridizing between nucleotide positions of genes 16S, msp2 and intergenic space 16S-23S, as indicated in tables 2 and 3. According to an even more preferred embodiment, the probes have sequences selected from the group SEQ ID NO:26-47, 48 and/or their complementary sequences. Hereinafter, these will be referred to as probes of the invention.

A fourth aspect of the invention relates to an analysis kit for the identification of any of the bacterial genera or species, as indicated in tables 2 and 3, where said kit comprises any of the triggers or probes of the invention. Additionally, this kit may include all the reactive agents, buffers, supports, etc. required for its development, without limitation.

TABLE 2 Detection and identification of species belonging to the genera Anaplasma/Ehrlichia ORGANISM Anaplasma (Ehrlichia) GENE TRIGGER PROBES SEQUENCES POSITION A. phagocytophilum msp2 SEQ ID NO: 18 MSP2 SEQ ID NO: 1 EF143812 (EF143812 (1-22)) (SEQ ID (223-243) SEQ ID NO: 19 NO: 26) (EF143812 (313-334) Generic 16S SEQ ID NO: 20. AEGEN SEQ ID U02521 (U02521 (9-30) (SEQ ID NO: 47 (38-57) SEQ ID NO: 21 NO: 48) (U02521 (109-86) Ehrlichia ruminantium 16S SEQ ID NO: 21 S-RUM SEQ ID NO: 2 DQ640401 SEQ ID NO: 21 (SEQ ID (23-45) NO: 27) E. sennetsu 16S SEQ ID NO: 20. S-SEN SEQ ID NO: 3 M73225 SEQ ID NO: 21 (SEQ ID (46-63) NO: 28) E. risticii 16S SEQ ID NO: 20. S-RIS SEQ ID NO: 4 AY005439 SEQ ID NO: 21 (SEQ ID (46-65) NO: 29) E. muris 16S SEQ ID NO: 20. S-MUR SEQ ID NO: 5 AY587608 SEQ ID NO: 21 (SEQ ID (16-37) NO: 30) A. platys 16S SEQ ID NO: 20. S-PLA SEQ ID NO: 6 EF139459 SEQ ID NO: 21 (SEQ ID (53-75) NO: 31) E. canis/E. ovina 16S SEQ ID NO: 20. S- SEQ ID NO: 7 EF011111 SEQ ID NO: 21 CANOVIN (14-37) (SEQ ID NO: 32)

TABLE 3 Detection and identification of species belonging to the genus Bartonella ORGANISM Bartonella GENE TRIGGER PROBES SEQUENCES POSITION Generic 16S SEQ ID NO: 22 BARTGEN2 SEQ ID NO: 8 AJ223780 (AJ223780 (SEQ ID (1054-1075) (961-979)) NO: 33) SEQ ID NO: 23 (AJ223780 (1376-1398)) B. talpae 16S-23S SEQ ID NO: 24 S-TOPO SEQ ID NO: 9 SEQ ID NO: (AY116638 (SEQ ID 43 (90-113) (455-478)) NO: 34) SEQ ID NO: 25 (724-743 (AJ269786)) B. phoceensis 16S-23S SEQ ID NO: 24 S-PHO SEQ ID AY515123 SEQ ID NO: 25 (SEQ ID NO: 10 (659-679) NO: 35) B. rattimasiliensis 16S-23S SEQ ID NO: 24 S-RAT SEQ ID AY515122 SEQ ID NO: 25 (SEQ ID NO: 11 (807-827) NO: 36) B. rochalimae 16S-23S SEQ ID NO: 24 S-ROC SEQ ID AF415211 SEQ ID NO: 25 (SEQ ID NO: 12 (414-434) NO: 37) Bartonella sp.* 16S-23S SEQ ID NO: 24 CHOSCA SEQ ID AY116639 SEQ ID NO: 25 (SEQ ID NO: 13 (438-461) NO: 38) Bartonella sp. 16S-23S SEQ ID NO: 24 S-APO38 SEQ ID AJ269792 in Apodemus SEQ ID NO: 25 (SEQ ID NO: 14 (425-445) sylvaticus NO: 39) Bartobella sp 16S-23S SEQ ID NO: 24 S-TEJ SEQ ID SEQ ID in Meles meles SEQ ID NO: 25 (SEQ ID NO: 15 NO: 44 (329-350) NO: 40) Bartonella sp. 16S-23S SEQ ID NO: 24 S-G-13 SEQ ID SEQ ID in Ixodes SEQ ID NO: 25 (SEQ ID NO: 16 NO: 45 ricinus 13 NO: 41) (92-111) Bartonella sp. 16S-23S SEQ ID NO: 24 S-G-41 SEQ ID SEQ ID in Ixodes SEQ ID NO: 25 (SEQ ID NO: 17 NO: 46 ricinus 41 NO: 42) (86-104) *B. chomeli/schoenbuchensis/capreoli/birtlesii

Brief explanation of Tables 2 and 3:

-   -   Column 1 (organism) indicates the bacterial species or group of         species detected in each case. The Bartonella sp. group refers         to a group of species belonging to this genus with a high degree         of similarity and which are jointly detected through a method of         the invention.     -   Column 2 (gene) indicates the gene or genome region used to         detect the bacterial species or group of species listed in         column 1.     -   Column 3 (trigger) indicates the sequence of the pair of         triggers required to amplify the variable regions of the gene or         intergenic space indicated in each table (column 2), in addition         to the sequence in which they hybridize.     -   Column 4 (probe) indicates the sequence of the probes used to         detect the bacterial species or groups of species listed in         column 1 of each table.     -   Column 5 (sequence 5′-3′) indicates the references of the         sequences of the variable regions which are amplified for the         detection of each bacterial species or group of species.     -   Column 6 indicates the sequence code of a gene region or genome         region listed in column 2, in addition to the specific position         of each sequence in which the probe indicated in column 4         hybridizes.

DETAILED DESCRIPTION

The present invention has allowed the development of an analysis method for the detection and identification of different bacterial genera and species using PCR or Multiple PCR technology. Methodology development required the analysis of intergenic space 16S-23S rRNA of genes 16S and msp2. These regions were analyzed combining different software applications and by comparison in databases, until the candidate regions susceptible to being used to apply the method were detected.

Said candidate regions were used to create a large number of triggers and probes, most of which, approximately 90%, were rejected after hybridization testing, until those which did not develop crossed reactivity with samples of different origin (FIGS. 1 and 2, Table 5) and, additionally, had high sensitivity levels, were finally selected.

Below is a detailed description of the materials and methods used in the development of the present invention, in addition to representative examples thereof. These examples do not limit the invention, but rather illustrate it, demonstrating the efficiency of the method of the invention. The use of these and other examples anywhere in the specification is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified form. Likewise, the invention is not limited to any particular preferred embodiments described herein. Indeed, modifications and variations of the invention may be apparent to those skilled in the art upon reading this specification, and can be made without departing from its spirit and scope. The invention is therefore to be limited only by the terms of the claims, along with the full scope of equivalents to which the claims are entitled.

EXAMPLES Amplification, Hybridization, and Validation

This step includes the experimental analysis of the variable regions detected earlier using PCR for their validation. The isolated DNA was amplified using PCR, applying the following temperature cycle table and reaction mixture composition, together with the specific triggers used previously for said purpose.

Temperature Cycles Temperature (° C.) Time Cycles 94  9′  1 94 15″

60  1′ 40 65  4′

65  7′  1

PCR reaction mixture composition for a final volume of 50 μL:

-   -   H₂O: According to final DNA volume     -   Buffer Taq Gold LD: 9 μL     -   Cl₂Mg [3 mM]: 6 μL     -   dNTPs [200 mM]: 1 μL×4     -   BSA [0.8 ug/uL]: 4 μL     -   14 specific Triggers (SEQ ID 1-2, SEQ ID 7-8, SEQ ID 25-26, SEQ         ID 28-29, SEQ ID 31-32, SEQ ID 36-37, SEQ ID 52-53) [50 pm/μL]:         0.5 μL of each (7 μL)     -   Taq Gold LD: 0.5 μL [2.5 units]     -   Problem DNA: maximum 800 ng

The amplicons were sequenced for their validation, verifying that the amplified sequence coincided with the variable sequences inferred from bioinformatic studies.

Subsequently, the amplicons were hybridized with specific probes according to the Reverse Line Blotting (RLB) protocol described by Sjoerd G. T. Rijpkema et al., Journal of Clinical Microbiology, December 1995, p. 3091-3095, although applying the following modifications (FIGS. 1 and 2):

-   -   Substrate: Super Signal West Dura (Pierce, Ref: 34075)     -   Probes: used with a concentration of between 0.2 and 3.2         picomoles/microlitre     -   Incubation: at 55LC     -   Lavages: at 52LC

Hybridization results are shown in FIGS. 1 and 2, where it is shown that each of the probes of the invention become joined specifically to each of the amplicons of the bacterial species detected using a method of the invention.

Preparation of Samples and Multiple PCR

One of the advantages of using PCR and RLB technology-based identification systems is that pure bacterial cultures are not required. In this manner and upon validation of the triggers and probes using DNA samples of the different species and subspecies listed in Tables 2 and 3, a Multiple PCR-based analysis of a DNA control mixture (FIGS. 1 and 2) prepared under laboratory conditions was carried out, followed by a RLB test, using the specifically designed triggers and probes and the previously indicated temperature cycles and reaction mixture composition.

Detection of PCR inhibitors

An IAC, which was amplified together with target DNA, was created for the detection of PCR inhibitors, using specific triggers (Table 4) designed according to the preserved regions of the AB183705 sequence belonging to the THC synthase gene of the Cannabis sativa species. Specifically, the IAC amplicon corresponds to a sequence of 371 pairs of bases, for which a probe was also designed for detection during RLB.

TABLE 4 IAC. Amplified gene, sequence of each of the triggers and probes used in the process and their relative position. POSITION ORGANISM GENE TRIGGER PROBES SEQUENCE 5′-3′ C. sativa THC Synthase SEQ ID NO: AB183705 51 (77-99) (CI-F) SEQ ID NO: AB183705 52 (CI-R) (447-427) C. sativa THC Synthase SEQ ID 51 SEQ ID NO: SEQ ID AB183705 (CI-F) 50 NO: 49 (281-302) SEQ ID NO: (S-CI2) 52 (CI-R)

Specificity of the Method

The high specificity of this method is based on the design and selection of the triggers and probes used, which were tested with another series of organisms (Table 5), following the previously described method, verifying that the formation of amplicons (FIGS. 1 and 2, right membrane) was in no case unspecifically detected.

TABLE 5 Specificity: unrelated bacterial, arthropod and mammal species used during method development. SPECIES RLB RESULT Bacteria 1 Brucella melitensis Negative 2 Chlamydia pneumoniae Negative 3 Chlamydia psittaci Negative 4 Legionella pneumophila Negative 5 Leptospira interrogans Negative 6 Mycoplasma pneumoniae Negative 7 Treponema pallidum Negative 8 Orientia tsutsugamushi Negative Arthropods Negative 9 Ixodes ricinus Negative 10 Rhipicephalus sanguineus Negative Mammals Negative 11 Apodemus sylvaticus Negative 12 Human Negative

All references cited and/or discussed in this specification are incorporated herein by reference in their entireties and to the same extent as if each reference was individually incorporated by reference. 

1. A method for detecting bacterial species that cause zoonosis, belonging to the genera Anaplasma/Ehrlichia and Bartonella, comprising the following steps: a. placing the sample under analysis in contact with a reaction mixture containing specific triggers that will amplify one or more sequences selected from the group comprising SEQ ID NO:1-17 and 47 or their respective complementary sequences; b. amplifying the sequences by means of polymerase chain reaction; and c. detecting amplification products formed in the previous step, said detection being indicative of the presence or absence of zoonosis-causing bacteria.
 2. The method according to claim 1, further comprising the step of identifying the bacterial species corresponding to the amplification products.
 3. The method of claim 2, wherein the steps of detecting and identifying are simultaneous.
 4. The method according to claim 1, wherein the triggers comprise sequences selected from SEQ ID NO:18-25 and their complementary sequences.
 5. The method according to claim 1, in which the detection of amplification products comprises using one or more probes having a sequence selected from SEQ ID NO:26-42 and 48 and their complementary sequences.
 6. A trigger having a sequence selected from SEQ ID NO:20-21 and their complementary sequences, said trigger allowing the amplification of a sequence selected from SEQ ID NO:2-7 and
 47. 7. The trigger of claim 6, wherein the trigger has a sequence selected from SEQ ID NO:22-23 and their complementary sequences, said trigger allowing the amplification of SEQ ID NO:8.
 8. A probe having a sequence selected from SEQ ID NO:26-42 and 48 and their complementary sequences, said probe being capable of hybridizing with a sequence selected from SEQ ID NO:1-17 and 47 and their complementary sequences.
 9. A kit for detecting bacterial species that cause zoonosis, belonging to the genera Anaplasma/Ehrlichia and Bartonella, comprising one or more triggers having a sequence selected from SEQ ID NO:18-25 and their complementary sequences.
 10. The kit according to claim 9, further comprising one or more probes having a sequence selected from SEQ ID NO:26-42 and 48 and their complementary sequences. 